Our long-term goal is to understand the molecular details and mechanisms involved in protein synthesis. Towards this goal, we will investigate the termination of protein synthesis in this proposal. The termination of protein synthesis occurs when the ribosome reaches the end of the coding sequence. It involves hydrolysis of the ester bond between the nascent peptide chain and P-site tRNA in the ribosome, with the resulting release of the peptide from the ribosome. Translational termination starts when a stop codon is encountered in the ribosomal A site and is recognized by protein class I release factors (RFs). These factors distinguish between sense codons and stop codons with very high accuracy, even without the proofreading that is the basis of tRNA selection for sense codons. The basis of how release factors recognize stop codons in the decoding center of the small subunit and transmit this recognition signal to catalyze peptide hydrolysis in the large subunit remains a major unsolved problem in translation. Class I RFs may induce a conformation of the ribosome that is activated for catalysis of peptide release. A detailed understanding of this fundamental process will require high resolution structures of the relevant functional complexes involved. Such structures will reveal molecular details of the interactions that occur during translational termination between the ribosome, P-site tRNA, class I RFs and their respective cognate stop codons. Hence, we propose (1) To determine high resolution crystal structures of the 70S ribosome in complex with class I RFs, cognate stop codons in the A site and non-hydrolyzable tRNA in the P site;this structure would represent the state just before peptide hydrolysis. (2) To determine high resolution crystal structures of the 70S ribosome in complex with class I RFs, cognate stop codons in the A site and deacylated tRNA in the P site;this would represent the state after peptide hydrolysis. (3) To use the structures determined in (1) and (2), or lower resolution structures already available, to mutate potentially important residues in RFs, tRNA or the ribosome and study their effects on RF binding to the ribosome, specificity of stop-codon recognition and catalysis of peptide release. PUBLIC HEALTH RELEVANCE: Translation is a fundamental process in all cells. Many viruses hijack the translation machinery by circumventing the normal translation pathway, and deregulation of translation is involved in various cancers. Thus detailed mechanistic studies of translation will be useful to understand these phenomena.